MicroRNAs contribute to critical brain functions and participate in multiple neurological and psychiatric conditions. Exactly how specific microRNAs relate to these processes remains unclear, however, for several reasons. First, genetic knockouts of brain-specific microRNAs have been difficult to generate. Second, predicting microRNA targets remains extraordinarily challenging. Third, methods for measuring microRNA levels in individual cells and, more importantly, linking these levels to the regulation of particuar targets are lacking. The overall objective of this application is to determine how the CREB-regulated microRNA, miR-132, directs dendritic growth and plasticity. Our specific aims are to: 1) Characterize the role of miR-132 in brain using a conditional knockout. We have already generated a mouse strain containing a floxed miR-132 allele and have shown, using a Cre-expressing retroviral vector, that newborn hippocampal neurons lacking miR-132 display a dramatic decrease in dendritic growth and branching. This was the first example of a neural-specific microRNA knockout, and we will use these mice to examine the role of miR-132 in adult neurogenesis, dendritic growth, and behaviors associated with activity-induced morphological changes, such as spatial learning and fear conditioning. 2) Determine whether heparin-binding (Hb)-EGF and the CDK inhibitor, p21, two miR-132 targets identified in Ago2-immunopurification assays, contribute to miR-132 effects on neuronal survival, morphology, and function. 3) Determine how changes in microRNA expression in individual neurons correlates with expression of specific microRNA targets. Assays of microRNA function typically average responses of large populations of cells. Using a new ratiometric microRNA sensor developed in our laboratory, we observed that individual neurons display large differences in the expression of miR-132. We will now ask whether these differences in miR-132 levels are associated with differences in target mRNA expression using, initially, cultured neurons and, subsequently, in vivo models. PUBLIC HEALTH RELEVANCE: MicroRNAs contribute to critical brain functions and participate in multiple neurological and psychiatric conditions, including Alzheimer's and Parkinson's diseases, schizophrenia, autism, and mental retardation. Exactly how specific microRNAs relate to these processes remains unclear, however. We propose a series of novel approaches to determine how a microRNA regulated by synaptic activity controls neuronal growth, function, and behavior by controlling the expression of specific growth and cell cycle regulators.